Despite the importance of sleep to our well-being and its impact on disease and work productivity, our cellular and molecular understanding of sleep regulation is limited. Sleep/wake regulation arises via the interaction between neurons. We are investigating the regulation of a sleep behavior during larval transitions called lethargus in the roundworm C. elegans. Lethargus shows both behavioral and molecular genetic similarities to sleep in mammals and Drosophila. Since identifying the neuronal circuit regulating sleep is the next step in the analysis of this behavior, we propose to identify sleep-regulating neurons in C. elegans. In Aim 1 we will conduct an optogenetic screen for interneurons that modulate feeding and locomotion rate, as sleep is characterized by cessation of these behaviors. In Aim 2 we will test whether candidate sleep-modulating interneurons identified in Aim 1 affect sleep-like sensory arousal thresholds and behavioral quiescence. We will also establish the order of action of sleep/wake active neurons. Our studies will make use of innovative optogenetic stimulation, optical imaging, and image processing technologies for perturbing neurons, quantifying feeding movements, measuring quiescence behavior, and measuring arousal thresholds. Our overall goal of identifying every interneuron modulating sleep/wake in an animal is a goal never before accomplished in any animal, yet it is feasible in C. elegans due to the simplicity of its nervous system. Our experiments will provide a knowledge base for mechanistic studies of genes affecting C. elegans sleep-like behavior as well as for physiological studies of sleep-regulating neurons.